Cells of the body are decorated with a variety of carbohydrates (sugars) that serve many diverse functions. These sugars not only act as a protective barrier on the outside of the cell, but are also involved in cell adhesion, migration, communication and signaling events in many organisms. Our group studies one type of sugar addition to proteins, known as mucin-type O-linked glycosylation, which is initiated by the polypeptide GalNAc transferase (ppGalNAcT or PGANT) enzyme family. This sugar addition is seen in most eukaryotic organisms including mammals, fish, insects, worms and some types of fungi. The conservation of this protein modification across species suggests that it plays crucial roles during many aspects of development. It is known that there are as many as 20 family members encoding functional ppGalNAcTs in mammals. Given the size of the family and the complexity it generates, we sought an alternative, simpler model system to investigate the biological role of glycosylation. Analysis of the genome databases from other organisms indicated that the fruit fly (Drosophila melanogaster) had only 12 potential members and may therefore be a more tractable experimental system. We began our studies by cloning and characterizing the genes responsible for O-linked glycosylation in Drosophila. We demonstrated that there are at least 9 functional transferase genes in Drosophila (potentially 12 members total) and that at least one is required for viability. These studies provided the first evidence that a member of this multigene family is required for development and viability in any eukaryote. Additionally, we defined the spatial and temporal patterns of expression of all the pgant family members throughout Drosophila development. We have also elucidated the developmental profile of specific O-glycans using a variety of sugar binding lectins and antibodies to determine where and when O-glycans may be required during development. Recently, we have found that mutations in another member of this family (pgant3) alter epithelial cell adhesion in the Drosophila wing blade. A transposon insertion mutation in pgant3 or RNA interference (RNAi) to pgant3 resulted in blistered wings, a phenotype characteristic of genes involved in integrin-mediated cell interactions. Precise excision of the transposon restored pgant3 expression and wing integrity. Mutations that form a stable yet inactive Golgi-localized enzyme also resulted in wing blistering, indicating that proper cell adhesion is dependent upon glycosyltransferase activity. Expression of wild type pgant3 in the mutant background rescued the wing blistering phenotype, whereas expression of another family member did not, revealing a unique requirement for pgant3 activity. Recently isolated point mutations in pgant3 show genetic interactions with an integrin mutant, demonstrating a genetic link between O-glycosylation and integrin-mediated cell adhesion. We have identified one of the main O-glycosylated proteins in the wing disc (tiggrin) using a combination of affinity purification, biochemistry and bioinformatics. Tiggrin is an integrin-binding extracellular matrix (ECM) protein that is specifically O-glycosylated in wild type wing discs but not in pgant3 mutants. We believe that pgant3 modulates integrin-mediated cell adhesion through glycosylation of this ECM protein. We are currently investigating the mechanistic role of O-glycans in this process. We are also investigating the role of each transferase using RNAi in vivo and in cell culture. This year, we have examined transgenic flies containing inducible dsRNA for each pgant family member. Expression of dsRNA has recapitulated the phenotypes described previously for pgant35A and pgant3, verifying the use of RNAi to specifically knockdown these genes in vivo. Interrogation of other isoforms by this approach indicates that additional pgants are required for viability. We are continuing this study by inducing RNAi to each family member under the control of various tissue-specific promoters to address the role of each isoform in certain developing tissues. We are following up these RNAi experiments with additional biochemical, cellular and genetic analyses of family members of interest. Finally, we are investigating the role of O-glycans in mammalian organ system development. Using salivary glands as our model system, we have assessed the expression levels of each of the 18 ppGalNAcTs during various stages of glandular development to determine their temporal patterns of expression. Our analyses demonstrate that certain isoforms are expressed at specific stages of glandular development. Additionally, there are a number of isoforms that are expressed tissue-specifically, in either the mesenchyme or the epithelium. To investigate the functional consequences of O-glycosylation, we have examined the growth and development of embryonic salivary glands from mice lacking one member of the ppGalNAcT family that is expressed most abundantly at early stages of development (E12-E13). While mice deficient in this transferase are viable and fertile, their salivary glands show a stage-specific developmental delay in growth. This stage-specific delay is likely the result of loss of specific protein glycosylation early in gland development. We are currently working to identify the glycoproteins that are expressed during early development to determine how they influence gland growth and morphogenesis. In summary, we are using information gleaned from Drosophila to better focus on crucial aspects of development affected by O-glycosylation in more complex mammalian systems. Our hope is that the cumulative results of the studies described above will elucidate why O-linked glycosylation is necessary and what role sugars play in cellular communication and interactions occurring during eukaryotic development.